Environment of tryptophan 57 in porcine fructose-1,6-bisphosphatase studied by time-resolved fluorescence and site-directed mutagenesis

Autor:	J, Wen, S W, Nelson, R B, Honzatko, H J, Fromm, J W, Petrich
Rok vydání:	2001
Předmět:	Models Molecular Aspartic Acid Protein Conformation Swine Circular Dichroism Tryptophan Crystallography X-Ray Recombinant Proteins Fructose-Bisphosphatase Kinetics Protein Subunits Spectrometry Fluorescence Amino Acid Substitution Mutagenesis Site-Directed Animals Asparagine
Zdroj:	Photochemistry and photobiology. 74(5)
ISSN:	0031-8655
Popis:	The environment of Trp57, introduced by the mutation of a tyrosine in the dynamic loop of porcine liver fructose-1,6-bisphosphatase (FBPase), was examined using time-resolved fluorescence and directed mutation. The Trp57 enzyme was studied previously by X-ray crystallography and steady-state fluorescence, the latter revealing an unexpected redshift in the wavelength of maximum fluorescence emission for the R-state conformer. The redshift was attributed to the negative charge of Asp127 in contact with the indole side chain of Trp57. Time-resolved fluorescence experiments here reveal an indole side chain less solvent exposed and more rigid in the R-state, than in the T-state of the enzyme, consistent with X-ray crystal structures. Replacement of Asp127 with an asparagine causes a 6 nm blueshift in the wavelength of maximum fluorescence emission for the R-state conformer, with little effect on the emission maximum of the T-state enzyme. The data here support the direct correspondence between X-ray crystal structures of FBPase and conformational states of the enzyme in solution, and provide a clear example of the influence of microenvironment on the fluorescence properties of tryptophan.
Databáze:	OpenAIRE
Externí odkaz:	https://explore.openaire.eu/search/publication?articleId=pmid________::359f413365fd5b9160b8eadf98a914e8 https://pubmed.ncbi.nlm.nih.gov/11723795 Zobrazit plný text záznamu Plný text